Fig. 2

T lymphocytes express ligand αvβ3 integrin and adhere to EGFL7. a Colocalization of CD4+ cells (green) and EGFL7 (red) in perivascular ECM area in MS brain lesions (upper panel = frontal, lower panel = temporal). Collagen IV = white. Nuclei (DAPI) = blue. Representative of n = 4 MS donors, ≥ 3 lesions/donor. b Expression of αvβ3 integrin on human CD14 monocytes ex vivo (left), memory CD4 T lymphocytes ex vivo (center), and in vitro activated memory CD4 T lymphocytes (right), as assessed by flow cytometry. Representative of n = 6 donors. c Expression of αvβ3 integrin on human CD4 T lymphocytes ex vivo and upon activation. Percentage and mean fluorescence intensity (MFI). n = 6 donors. d Expression of αvβ3 integrin on human CD4 T lymphocytes ex vivo from healthy controls, untreated MS or treated MS. Flow cytometry analysis. 1 dot = 1 donor. e, f Expression of αvβ3 integrin by murine CD4 T lymphocytes (e) ex vivo and on day 1–5 in vitro upon activation, and (f) in naïve and MOG_35–55-immunized (EAE) mice at different time points of EAE. n = 4–5 mice/condition, 1 dot = 1 mouse. g–k Adhesion assay. g Representative microscopy images of CFSE-labeled activated human CD4 T lymphocytes adhering to rhEGFL7 or BSA 1% after 24 h. Scale bar = 100 μm. Amount of human (h) and murine (j) activated CFSE-labeled CD4 T cells adhering to coated rh or rmEGFL7, ICAM-1 or fibronectin, normalized to BSA 1%, according to fluorescence intensity as assessed by plate-reader. n = 6 human donors and n = 8 mice. Amount of human (i) and murine (k) cells adhering to coated EGFL7, ICAM-1 or fibronectin, following pre-treatment with rhEGFL7 (i) or rmEGFL7 (k); anti-αvβ3 integrin neutralizing antibody (i) or RGD-peptide (k); or relevant control (rat serum, isotype or RAD-peptide). n = 7 human donors and n = 8 mice. Statistical analysis performed by Wilcoxon matched-pairs signed rank test (c) and one-way ANOVA followed by Tukey’s multiple comparison test (d–k). *p < 0.05, **p < 0.01, ***p < 0.001. All data presented as mean ± SEM